The Plastisphere - Uncovering tightly attached plastic "specific" microorganisms

Abstract

In order to understand the degradation potential of plastics in the marine environment, microorganisms that preferentially colonize and interact with plastic surfaces, as opposed to generalists potentially colonising everything, need to be identified. Accordingly, it was hypothesized that i.) plastic "specific" microorganisms are closely attached to the polymeric surface and ii.) that specificity of plastics biofilms are rather related to members of the rare biosphere. To answer these hypotheses, a three phased experiment to stepwise uncover closely attached microbes was conducted. In Phase 1, nine chemically distinct plastic films and glass were incubated in situ for 21 months in a seawater flow through system. In Phase 2, a high-pressure water jet treatment technique was used to remove the upper biofilm layers to further, in Phase 3, enrich a plastic "specific" community. To proof whether microbes colonizing different plastics are distinct from each other and from other inert hard substrates, the bacterial communities of these different substrates were analysed using 16S rRNA gene tag sequencing. Our findings indicate that tightly attached microorganisms account to the rare biosphere and suggest the presence of plastic "specific" microorganisms/assemblages which could benefit from the given plastic properties or at least grow under limited carbon resources.

Fig 1. Experimental design.

Schematic presentation of the three phased stepwise uncovering experiment of potential plastic “specific” bacteria.

Fig 2

High-pressure water Jet treatment with the a) high pressure treatment device. b) Barplot of the enumerated mean of adherent membrane intact (green) and membrane damaged (red) cells after a high pressure treatment at 4 bar for 2 minutes, vertical bars denote the Standard Error. Photograph of the 21 month old biofilm attached to c) Polylactic acid and d) Low density polyethylene. Resulting spots e; f) in respective biofilms after high pressure treatment. Double stained (SYBR Green & PI) cells on respective substrate g; h) after high pressure treatment with, scale bars are 10 μm.

Fig 3

SEM images of colonized plastics. a) Meshwork of morphological diverse cells embedded in EPS attached to PS. b) Colony attached to PESTUR c) Single cells of rods and cocci on HDPE d) Consortia of rods and cocci embedded in EPS on PS e) Rod with spore, comma and spiral cells on PVC.

Fig 4. Richness of the bacterial communities attached to the diverse substrates based on the number of observed OTUs.

Vertical bars denote the standard deviation (n_short-term = 1; n_long-term = 5).

Fig 5. Principle Coordinate Ordination relating variation in the community composition between different short- and long-term incubated substrates.

PCOs representing similarity of biofilm communities based on relative abundances of OTUs across samples. OTUs with a mean relative abundance of at least 0.1% in one substrate type (n_short = 1; n_long = 5) were analysed. The different colours indicate the respective substrate, filled symbols represent short-term samples, open symbols long-term samples. Arrows connect short- and long-term samples of the respective substrate.

Fig 6. Biofilm community composition based on abundance profiles of the short- and long-term communities on the class level on different plastics and glass.

OTUs with a mean relative abundance of at least 0.1% in one substrate type (n_short = 1; n_long = 5) were analysed. A * indicates the term “unclassified”, a # indicates the term “Incertae Sedis”.

Fig 7. Shade Plot of plastic “specific” OTUs (indicated by numbers) on different long-term plastics and comparison of their relative abundance in untreated mature biofilms of the same experimental set up after 15 month.

Abundant OTUs (mean relative abundance <0.1%; n = 50) are indicated in turquoise, rather rare OTUs (mean relative abundance >0.1%; n = 50) are indicated in black. Shade Plot creation was based on square root transformed relative abundances. OTUs with a mean relative abundance of at least 0.1% in one substrate type (n = 5) were analysed. Displayed are OTUs jointly contributing, with a minimum of 3%, to the total dissimilarity between different plastic groups (SIMPER analysis). OTUs with a mean relative abundance of at least 0.1% present on both, plastics and glass, were rejected. The amount of contribution is indicated by the colour of cells, lighter colours represent higher contributions. A * indicates the term “unclassified”, # indicates the term “uncultured”.